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 JoVE General

Establishing Primary Adult Fibroblast Cultures From Rodents

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1Department of Biology, University of Rochester

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    Summary

    This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

    Date Published: 10/05/2010, Issue 44; doi: 10.3791/2033

    Cite this Article

    Seluanov, A., Vaidya, A., Gorbunova, V. Establishing Primary Adult Fibroblast Cultures From Rodents. J. Vis. Exp. (44), e2033, doi:10.3791/2033 (2010).

    Abstract

    The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

    Protocol

    1. Before Starting

    1. Sterilize scissors and forceps with 70% ethanol.
    2. Place small magnetic stirrer inside a 30 mL beaker, cover with two layers of foil, and autoclave.
    3. Prepare a 28 Wunsch units/mL stock solution of Liberase Blendzyme 3 in sterile water. Make 0.5 mL aliquots and freeze at -20°C Thaw a new aliquot before every use. The solution may appear cloudy after thawing. Vortex the solution until it becomes clear.
    4. Warm up the cell culture media.

    2. Animal Sample Preparation

    Caution: wild animals may contain pathogens such as a rabies virus. Always be aware of open sharps.

    1. Euthanize the animal and place the carcass at 4°C. It is best to use the carcass immediately, however, if this is not practical such as if the animals are collected in the field, the carcasses may be used within 24 hours. After 24 hours the cell yield declines.
    2. Dissect the animal in the surgical suite or in chemical hood (not in tissue culture hood).
    3. Underarm area is a convenient site to collect skin samples as underarm skin is thinner, contains less fat, and the fur is less dense. Clean the incision site with 70% ethanol. Make sure the fur is soaked with ethanol.
    4. Shave the fur around the incision site with a sharp scalpel. Shave a larger area than the desired incision site. Try to minimize cuts to the skin. Spray the area with 70% ethanol and let it dry.
    5. To collect a skin sample excise a fragment of approximately 1 cm2. Pinch the skin with tissue forceps, and cut with scissors. Try not to cut the fat layer with the skin, as fat interferes with collagenase digestion. To collect the lung samples, wash the chest area with 70% ethanol, and open the skin on the chest with scissors by making a T-shape incision and pulling apart the flaps of the skin. Wash the opened muscle area with 70% ethanol and let dry. Cut the rib cage using sterile forceps and scissors (use bone cutters for larger animals). Use sterile techniques to avoid contaminating internal organs. Do not touch the internal organs with instruments that touched the animal fur. Cut lung fragments of approximately 1 cm2 using sterile forceps and scissors.
    6. Place tissue fragments in 50 mL tubes with sterile PBS to avoid drying.
    7. Wash the outside of the 50 mL tubes with tissue fragments with ethanol, and take them to the tissue culture hood.
    8. Properly dispose of animal carcass.

    3. Extracting Cells

    1. Transfer the tissue fragments into a 10 cm tissue culture dish using sterile scalpel. Do not transfer too much PBS with the sample.
    2. Cut the tissue into ~1 mm pieces using two scalpels. Use two blades using a scissor action starting from the center and pulling apart. Keep the tissue balled up, do not cut piece by piece. When the cutting is sufficient the skin resembles putty, it will not separate into pieces but will stretch thin. The lung tissue is easier to cut, and it will separate into tiny pieces.
    3. Using a scalpel, transfer the cut tissue into sterile 30 mL beaker with sterile stir bar. Wash the plate used for cutting tissue with 10 mL of DMEM/F12 media with 0.14 Wunsch units/mL Liberase Blendzyme 3, and 1X antibiotic/antimycotic, and add the solution to the 30 mL beaker with tissue fragments.
    4. Cover the beaker with sterile foil, and incubate at 37°C, stirring slowly, for 30 to 90 minutes. The length of the incubation depends on the species and tissue type. Take care not to over-digest the tissue. Best yields are obtained when tissue fragments are still present at the end of the digestion. Skin takes longer to digest than the lung. Skin from large animals takes longer to digest. Check the digestion after 30 min, and then every 10 min. When the skin digestion is complete, the media becomes cloudy and skin fragments separate from each other, and the edges of the pieces become "fuzzy". Lung digestion should be stopped when lung fragments change color from red to white and start forming sticky fibers.
    5. Pipet the solution containing tissue fragments up and down to break the clumps. Transfer the solution to sterile 50 mL tube. If the fragments move easily through the 10 mL pipette cutting and digestion was done well. Rinse the beaker 3 times with 10 mL of warm DMEM/F12 media with 15% FBS, 1X antibiotic/antimycotic and add the media to the 50 mL tube with tissue fragments. Close the 50 mL tube and mix by inversion a few times. The FBS in the media will stop Liberase digestion.
    6. Spin at 524 g in a swinging bucket tissue culture centrifuge for 5 min. Remove the supernatant Resuspend the pellet in 10 mL of warm DMEM/F12 media with 15% FBS, 1X antibiotic/antimycotic. Pipet the suspension with maximum force to break the tissue pieces.
    7. Add another 30 mL of DMEM/F12 media with 15% FBS, 1X antibiotic/antimycotic, mix and centrifuge at 524 g in a swinging bucket tissue culture centrifuge for 5 min. Repeat one more time to remove the traces of Liberase.
    8. Resuspend the pellet in 10 mL of DMEM/F12 media with 15% FBS, 1X antibiotic/antimycotic and transfer to a 10 cm tissue culture dish and place in a tissue culture incubator at 37°C, 5%CO2, 3%O2.
    9. Check the plates every day for fibroblasts and media color. If the isolation was successful, fibroblasts crawl out of the tissue fragments and attach to the plate (Figure 1). The fibroblast start to exit tissue fragments within 2-5 days.
    10. If the media changes color to yellow, this indicates potential contamination or overcrowding of cells. Examine the plates under the microscope at high magnification If bacteria, fungi, or worms are present discard the plates (this is rarely an issue with laboratory animals, however may occur when the samples are collected from the wild). If no contamination is present, but the media changed color, this is caused by either too many cells or too many tissue fragments placed in the same dish. If more than 60% of the plate is covered by attached fibroblasts, change the media on the plate and transfer the tissue fragments to a new plate with the new media. If not many fibroblasts attached to the plate, change the media and split the tissue pieces to a 2-4 plates.
    11. After 7 days, if media had not been changed earlier, change the media and transfer the tissue fragments to a new plate with new media.
    12. Incubate the cells and tissue fragments for an additional 7 days. By day 14 all viable fibroblasts have exited the tissue fragments.
    13. After 14 days from the beginning of the cell isolation, discard the old media and tissue fragments, harvest the cells and plate them on a new plate at 5x105 cells/plate EMEM with 15% FBS, 1X Penicillin/Streptomycin, non essential amino acids, and sodium pyruvate. EMEM media will support growth of fibroblasts only and other cell types will die or stop proliferating.
    14. After the cells reach 80-90% confluence, freeze an aliquot of cells for future use.
    15. Continue culturing the cells by splitting them at 5x105 cells/plate when the cells reach 80-90% confluence.

    4. Representative Results

    Normal fibroblasts are large cells with prominent protrusions (lamellipodia) (Figure 2). Fibroblasts grow in a monolayer. A healthy growing culture contains 1-10% cells in M stage, recognized as rounded up cells elevated over the surface of the plate, but not detached from the plate (Figure 3). Typically, a 10 cm dish seeded with 5x105 cells becomes confluent in 3-4 days. The doubling time varies greatly between species, and may be greater for some of the long-lived rodent species1. When cells fill a plate they arrest proliferation in G1 stage. Typical confluent plate of fibroblasts contains a tightly packed layer of cells (Figure 4). When the cells reach 90% confluence they are ready for splitting. If desired, the cells can be maintained on an arrested confluent plate for extended periods of time with regular media changes (once or twice a week).

    Figure 1
    Figure 1. Fibroblast isolation from mouse skin (A) and lung (B). The media was changed to remove unattached cells and debris prior to taking the pictures on Day 7.

    Figure 2
    Figure 2. Mouse lung fibroblasts.

    Figure 3
    Figure 3. A field of mouse fibroblasts containing cells in M-stage, photographed at 10X magnification.

    Figure 4
    Figure 4. A confluent plate of mouse fibroblasts, photographed at 10X magnification.

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    Discussion

    Normal primary fibroblasts provide an excellent alternative to established cancer cell lines in biological research. An important advantage of fibroblasts is that they do not carry mutations in oncogenes and tumor suppressors and maintain intact cell cycle checkpoints. This makes normal fibroblasts a preferred system for the studies of cell cycle regulation, DNA repair, and apoptosis. The protocol described here provides a simple recipe for isolation and maintenance of primary fibroblasts. This protocol was used to successfully isolate fibroblasts from over 20 species of rodents.

    It is important to maintain sterility at all times when working with cell cultures to avoid contamination. If the laboratory also cultures aggressive cancer cell lines such as HeLa cells, fibroblasts should be handled separately from cancer cells. It is preferred to have a designated hood and incubator for primary cultures.

    It is preferable to maintain primary cell cultures at physiological oxygen concentration of 3%. Atmospheric (20%) oxygen shortens lifespan of cultures and increases oxidative stress. Mouse fibroblasts are sensitive to oxidative stress and will senesce or enter crisis within ~14 population doublings when maintained at 20% (atmospheric) oxygen. Mouse fibroblasts can be maintained indefinitely at 3% oxygen2.

    Always keep record of the number of population doubling of the cultures. Large species (body mass above 8,000 g) are likely to exhibit replicative senescence, and will have a limited lifespan in culture, even at 3% oxygen1. Cells from smaller species, such as mice and rats, will proliferate indefinitely at 3% oxygen, but may eventually become aneuploid. If possible, begin the experiments using cells at low population doubling.

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    Disclosures

    No conflicts of interest declared.

    Acknowledgements

    We thank Dr. Steven Austad for providing us with the first version of this protocol. This work was supported by grants from the NIH and the Ellison Medical Foundation to V.G. and A.S.

    Materials

    Name Company Catalog Number Comments
    DMEM/F12 media Invitrogen 11330-032
    Fetal Bovine Serum (FBS), Qualified Invitrogen 01437-036 The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.
    Antibiotic/Antimycotic Invitrogen 15420-096
    Penicillin/Streptomycin Invitrogen 15140-122
    Liberase TM Research Grade Roche 05401127001 Replacement enzyme.
    A note from the authors: Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.
    EMEM media ATCC 30-203 The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.
    Feathered #21 disposable, sterile scalpel Multiple suppliers
    Three Gas Control incubator Forma or Heraeus

    References

    1. Seluanov, A. et al. Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging Cell 7, 813-23 (2008).
    2. Parrinello, S. et al. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 5, 741-7 (2003).

    Comments

    28 Comments

    Very helpful protocol. Got it working at the very first try, Would highly recommend the protocol.
    Reply

    Posted by: AnonymousFebruary 14, 2012, 5:10 PM

    I have recently isolated fibroblasts from fresh fibrotic human lung. I used collagenase and plated the cells via your protocol. the cells seem to be of ² populations: one is long (²0% of plate) typical of fibroblast but the other cells are round. They look like they just started to adhere to the plate. What cells could these be? Is this happen often? COuld they be a subset of fibroblasts?
    Reply

    Posted by: Barbara S.April 4, 2012, 9:33 AM

    Hi
    The round cells are epithelial cells that always accompany lung fibroblast isolation. These cells will just float. After you switch the media to EMEM (mentioned in Step 13) and start splitting the fibroblasts, these cells will go away.
    Reply

    Posted by: AnonymousApril 5, 2012, 12:11 PM

    I have used your protocol successfully before on human lung tissue. However this time, the majority of our cells were epithelial- look very much like keritanocytes. Is there something we could do to only keep the fibroblasts growing- change to MEM media?
    Also, the next time we try this protocol, is there anything we can do to not get so many epithelial cells?WIll be happy for any suggestions. Thanks.
    Reply

    Posted by: Barbara S.April 5, 2012, 8:14 AM

    Hi Barbara,
    We always get the round epithelial cells during our lung fibroblast isolation too. When you switch to EMEM media and start splitting the fibroblasts, they will be selected for and the epithelial cells will be lost.
    Next time you try the protocol, you can use EMEM media for the whole isolation procedure instead of DMEM/F1² to minimize the epithelial cells you get. But the trade-off is that the efficiency of isolation might decrease. Good luck.
    Reply

    Posted by: AnonymousApril 5, 2012, 12:15 PM

    A note from the authors:
    Since Roche discontinued Liberase Blendzyme 3, they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 054011²7001 - 100mg) instead. We have switched to this enzyme successfully with no issues.
    Reply

    Posted by: AnonymousApril 5, 2012, 12:31 PM

    Thank you so much for your reply. I split them yesterday and changed the media to MEM.
    Reply

    Posted by: Barbara S.April 6, 2012, 7:35 AM

    What is the best way to remove tissue peices on day 7? Especially with the larger tissue peices, ones that you can clearly see, produce more epithelial cells, where as the small peices (as in your pictures) produce mostly pure fibroblasts. Also, do you transplant the tissues on day 7 regardless of the confluency of your plate?
    Reply

    Posted by: AnonymousApril 6, 2012, 11:59 AM

    On day 7, we gently aspirate the supernatant (with the tissue pieces) with a pipette, transfer it to a new plate and provide the original plate with fresh media. In this way, the small pieces attached to the plate are not disturbed and the other tissue debris with the potential to give out more fibroblasts can do so on a new plate.
    Also, yes, it is a good idea to transfer the tissues on a new plate on day 7.
    Reply

    Posted by: AnonymousApril 6, 2012, 3:43 PM

    When you trypsinize initially, do you do this aggressively and try to remove all the cells from the plate (fibroblast and epithelial)? I have found that the fibroblasts seem to trypsinize first and can thus help to purify the culture, but my cell yield is significantly smaller (too small). Thank you for you help.
    Reply

    Posted by: AnonymousApril 6, 2012, 4:05 PM

    The first trypsinization dŒs not have to be aggressive. As long as all the fibroblasts are off the plate and floating, we dont worry about the epithelial cells. They usually disappear after the first couple of passages with EMEM media. The fibroblasts are fast growing and will be selected for with passaging.
    To improve the yield, I would suggest ² things in the isolation procedure : Mincing the tissue very well and incubating the tissue pieces for a longer time in the Collagenase enzyme (while taking care to avoid over-digestion).
    Reply

    Posted by: AnonymousApril 6, 2012, 4:18 PM

    You recommend storing 0.5 mL aliquots of Blendzyme at ²8 units/ml (i.e. 14 wunsch units per aliquot) but you recommend using 0.14 units/ml in 10 mls of media, or 1.4 wunsch units per digestion) This implies each aliquot is for 10 digestions. Is this correct?
    Reply

    Posted by: AnonymousApril 16, 2012, 10:59 AM

    Yes
    Reply

    Posted by: AnonymousApril 16, 2012, 11:04 AM

    After 14 days I achieve confluence and then split into fibroblast specific growth media and these cells have no trouble reaching 80-90%. When I subsequently split this population the cells senesce. Is there any obvious reason for this? It would think that 14 days of growth would cause a primary cell to senesce but your protocol suggests that I should be able to continue splitting my cells and further purfy in the process. I appreciate your help!
    Reply

    Posted by: Justin K.May 4, 2012, 10:49 AM

    Hi Justin
    Are these mouse fibroblasts? Are you growing them in ²0% oxygen? If you are, then they will senesce. We grow all our cells in 3% oxygen and 5% CO² conditions.
    Reply

    Posted by: Amita V.May 9, 2012, 3:06 PM

    Hi, one of the first steps recommends to euthanize the animal and store it at 4oC. Do we complete this step even if we plan on using the animal right away? If so, for how long do we store the animal at 4oC? Is putting it on ice also acceptable?
    Reply

    Posted by: J A.May 13, 2012, 7:49 PM

    No..It is best to use the animal right away. The 4C step is only if circumstances make it impossible to process it right away. In this case, we store the corpse at 4C(better) or on ice(in case of wild caught animals) for not more than ²4 hours.
    Reply

    Posted by: Amita V.May 13, 2012, 7:57 PM

    Also, another question I have is about the tissue fragments in the media. When would be the best time to remove them from the cells and replace them with media?
    Reply

    Posted by: AnonymousMay 14, 2012, 12:42 AM

    It depends. Usually, if the media turns yellow a few days after isolation and if there aren't enough fibroblasts on the plate, we collect the fragments, suspend them in fresh media and plate them back. As the protocol says, day 14 is when we usually get rid of the tissue fragments.
    Reply

    Posted by: Amita V.May 14, 2012, 2:51 PM

    Hi,

    I a couple of questions about the reagents used. My first question is about the liberase enzyme used. I noticed that the liberase enzyme 3 from Roche has been discontinued. Would you recommend replacing it with the Liberase TM Research Grade medium enzyme with Thermolysin? If so, would you recommend keeping the concentration the same (.14 U/mL)? My second question is about the Fetal Bovine Serum used in this experiment. I understand that you recommend using the Qualified Fetal Bovine Serum because it has been shown to support the growth of primary fibroblast cultures. In my lab we use heat-inactivated FBS. Would you recommend me switching from heat-inactivated to qualified FBS?

    Thank you in advance for your help :)
    Reply

    Posted by: J A.May 21, 2012, 11:25 AM

    Yes. We now use TM instead of Liberase 3 with the same concentration.
    I understand that heat-inactivation of FBS is usually done to get rid of complement factors. I don't know how critical it is for your experiments to do this but we never use heat-inactivated serum.
    Reply

    Posted by: Amita V.May 24, 2012, 3:06 PM

    I also have a question about plating the tissue into the media. ²4- hours after I plated my tissue pieces into the media, I looked at my cultures under the microscope and saw little round cells that adhered to the plate. I understand that the protocol mentions that fibroblasts do not typically exit the tissue for at least 48 hours, and the round epithelial cells that assist the growth of fibroblasts usually float through the media. I was just wondering if seeing these cells after ²4 hours is normal.
    Reply

    Posted by: J A.May 24, 2012, 12:59 PM

    Yes it is normal, more for lung fibroblast isolation. Once the fibroblasts start growing, the round cells will be selected against.
    Reply

    Posted by: Amita V.May 24, 2012, 2:57 PM

    Hi,

    I was wondering what concentration of trypsin EDTA you used? I use trypsin 0.²5% trypsin EDTA to remove my cells during splitting (after an incubation time of 4 minutes). After trypsinizing the cells for 4 minutes, I have noticed that less than half of them are detached from the plate. I am afraid to leave the cells in trypsin for longer than 4 minutes because I do not want to damage them. Any suggestions?
    Reply

    Posted by: J A.July 18, 2012, 1:05 AM

    We use Red trypsin with 0.²5% EDTA..Its very strong and we only treat cells with it for 1-² minutes. So you wash/rinse the plate with PBS after aspirating media, before adding trypsin? Because the FBS in the media inhibits trypsin activity and if the plates are not washed with PBS first, trypsin cannot act properly.
    Reply

    Posted by: Amita V.July 18, 2012, 11:28 AM

    Thank you for such detailed procedure. I am curious is it possible that the keratinocytes also exist in the fibroblast culture? Thank you.
    Reply

    Posted by: Hua-Ling C.September 19, 2012, 3:16 AM

    Dear all, my adult fibroblasts (GFP positive C67Bl/6 mice) are not growing very well (DMEM, high glucose, Na-pyrovate, Pen/Strp, 15%FBS). Do you have any suggestions about what I could add to the media to increase proliferation? Thanks a lot. Gunnar
    Reply

    Posted by: Gunnar P.March 1, 2013, 2:03 PM

    Can this protocol be adapted to isolate cancer-associated fibroblasts?
    Reply

    Posted by: Wei L.July 9, 2013, 2:59 PM

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